A few importers of marine products has practiced ice glazing of frozen fish and forced water injection into small octopus to increase their weight. These rampant practices have recently become a serious social issue. Therefore, this study was conducted to develop non-destructive detection methods for verifying forced water injected frozen small octopus using dielectric properties. The weight and dielectric properties of live small octopuses imported from China were measured using an electronic scale and dielectric probe connected with vector network analyzer, respectively. The frequency range from 0.5 to 4 GHz was used for measurement of dielectric properties of small octopus samples. The moisture contents of live small octopuses were determined by convection drying at 105°C for 24 h. To increase weight of small octopus samples, each sample was placed in a container including 2% polyphosphate solution of 800 mL and was kept for 24 h in a refrigerator. Then, the sample was removed from the solution and was frozen at -35°C for 24 h. The moisture contents of live small octopuses were 81.6 ± 1.9% (wb). Regardless of weight of live small octopuses, dielectric constant (ε′) and dielectric loss factor (ε′′) were from 76.09 to 58.62 and from 101.95 to 28.72, respectively. The average weight gain of small octopuses immersed in the solution was approximately 40.6%. Dielectric constant (ε′) and dielectric loss factor (ε′′) of forced water injected small were from 78.18 to 66.71 and from 51.96 to 25.05, respectively. In addition, there was a significant difference between dielectric properties and penetration depth of fresh frozen and forced water injected frozen small octopuses. The results clearly showed that dielectric properties of small octopuses affected by moisture contents could be an important factor to detect forced water injected frozen small octopuses.

The objective of this research was to evaluate the effects of two different feeding systems on blood metabolites in Holstein heifers and analyze the economic impacts of the feeding systems. The following two experiments were conducted to investigate the effects of feeding system on blood metabolite changes in Holstein heifers and analyze the economic impacts of the two systems. In experiment 1, the effects of two different feeding systems on cortisol, progesterone, and estradiol in Holstein heifers were examined. In experiment 2, the effects of two different feeding systems on the body weights of Holstein heifers and profitability of the two feeding systems were studied. Results showed that the pasture-raised heifers showed significantly decrease in the levels of blood cortisol (p<0.05) and increases in the levels of progesterone and estradiol (p>0.05) when compared with heifers raised in indoor feeding system. The average daily gain was significantly higher (p<0.05) in indoor-raised heifers (0.73 kg/day) as compared to pasture-raised heifers (0.58 kg/day). Also, 25.2% more profits were obtained from the pasture feeding system as compared to the indoor feeding system. These results together would be useful in the investigation of feeding system and growth performance in dairy cattle.

The habitat of the cockroach varies by species. The German cockroach (Blattella germanica) lives in human dwellings, while the Japanese field roach (Blattella nipponica) lives in a mountainous region. Based on phylogenetic analysis of mtCO I, the two species are closely related to each other and B. germanica is divergent from wild species such as B. nipponica. Their habitats and walking speed differ even though the two species have similar morphology. We hypothesized that habitats might influence walking speed via changes to appendage morphology and enzyme-based physiological differences. We found that phenotype such as appendage length and esterase isozyme expression were clearly different between the two species. These differences might be responsible for the observed difference in walking speed.

Pigmentation or melanization is an important physiological event in insects and is involved in cuticle tanning, wound healing and encapsulation as a defensive response. Dopachrome-conversion enzyme (DCE, Yellow) significantly accelerates the melanization of the chorion in mosquito eggs. In this study, we demonstrated functional importance of two ovary-specific yellow genes, AalY-g and AalY-g2, in the Asian tiger mosquito, Aedes albopictus. The transcripts of both genes were detected in the ovary of adult females only 48-72 h after blood feeding. RNAi for AalY-g or AalY-g2 had no effect on fecundity. However, the outermost colorless exochorion of the eggs was fragile and partially peeled off, and initial melanization of the endochorion was significantly delayed. Eggs from control females exhibited high desiccation resistance, whereas those from dsAalY-g- or dsAalY-g2-treated females were collapsed (50-80%) under the air-dry condition. Ultrastructural analysis revealed abnormal morphology of the endochorion and vitelline membrane in the AalY-g and AalY-g2-deficient eggshell. These results indicate that AalY-g and AalY-g2 are critical for integrity and desiccation resistance of the Ae. albopictus eggs. This work was supported by NRFs (NRF-2015R1A6A3A04060323).

The Riptortus (stinkbug) has a specialized symbiotic organ, M4 midgut, to harboring symbiont Burkholderia. M4 midgut is located in abdomen and surrounded with insect hemolymph. Recently our group demonstrated that symbiotic Burkholderia showed different physiology after adapting in M4 gut compare with in vitro cultured Burkholderia. And population of symbiotic Burkholderia in the M4 midgut is regulated by special organ. However, the molecular mechanism to prevent spreading and migrating symbiont bacteria to other host tissues from symbiotic organ is not clear. Therefore, we assumed that symbiont Burkholderia are susceptible to host humoral immunity after established infection in M4 midgut to prevent spreading and migrating into the other host tissues through Riptortus hemolymph. To prove this assuming, we tested the susceptibility and survival rate of symbiont Burkholderia in hemolymph of Riptortus in vitro and in vivo. We also examined the susceptibility of symbiont Burkholderia using purified antimicrobial peptides (AMP), pyrrhocoricin-like, thanatin-like and defensin-like AMPs. Finally, we tested inducing ability for AMPs by systemic infection of symbiotic Burkholderia. Gene expression of purified AMPs was not different after systemic infection of both symbiont and in vitro cultured Burkholderia. Surprisingly, in vitro cultured Burkholderia resisted on bacteria injected hemolymph and purified AMPs but symbiont Burkholderia were highly susceptible in bacteria injected hemolymph and purified AMP. These results suggest that symbiont Burkholderia can't survive in the hemolymph after escaping symbiotic organ. Moreover, humoral immunity of host Riptortus is important to prevent spreading and migrating symbiont Burkholderia into the other host tissue or organ from symbiotic organ.

Most traditional genome sequencing projects involving infectious viruses include culturing and purification of the virus. This can present difficulties as an analysis of multiple populations from multiple locations may be required to acquire sufficient amount of high-quality DNA for sequence analysis. The electrophoretic method provides a strategy whereby the genomic DNA sequences of the Korean isolate of Pieris rapae granulovirus (PiraGV-K) were analyzed by purifying it from host DNA by pulsed-field gel electrophoresis, thus simplifying sampling and labor time. The genomic DNA of infected P. rapae was embedded in agarose plugs, digested with a restriction nuclease and methylase, and pulsed-field gel electrophoresis (PFGE) was used to separate PiraGV-K DNA from the DNA of P. rapae, followed by mapping of fosmid clones of the separated viral DNA. The double-stranded circular genome of PiraGV-K encodes 120 open reading frames (ORFs), covering 92% of the sequenced genome. BLAST and ORF arrangement showed the presence of 78 homologs to other genes in the database. The mean overall amino acid identity of PiraGV-K ORFs was highest with the Chinese isolate of PiraGV (~99%), followed up with Choristoneura occidentalis ORFs at 58%. PiraGV-K ORFs were grouped, according to function, into 10 genes involved in transcription, 11 involved in replication, 25 structural protein genes, and 15 auxiliary genes. Genes for Chitinase (ORF 10) and cathepsin (ORF11), involved in the liquefaction of the host, were found in the genome. The recovery of PiraGV-K DNA genome by pulse-field electrophoretic separation from host genomic DNA had several advantages, compared with its isolation from particles harvested as virions or inclusions from the P. rapae host. We have sequenced and analyzed the 108,658 bp PiraGV-K genome purified by the pulsed field electrophoretic method. The method appears to be applicable to the analysis of genomes of large viruses. The chitinase, identified by PiraGV-K genome sequence, was functionally characterized by quantitative PCR, Western blot analysis, immunohistochemistry and transmission electron microscopy.

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146 Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5’-flanking region. BLAST and phylogenetic analysis reveals that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic helices, including a short helix 3’. The ‘helix-short helix-helix’ motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly up-regulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor.

Entomopathogenic fungi are natural pathogens of insects and contribute to the regulation of host insect populations in the environment. Several these fungi produce a wide range of secreted enzymes, secreted protein toxins and secondary metabolites to overcome host defenses and ultimately kill the host, and to defend host resources against competing pathogens and saprophytes. Therefore, this study was performed to select the antimicrobial activity of entomopathogenic fungi form Korea soils against plant pathogenic bacteria Ralstonia solanacearum and plant pathogenic fungi Botrytis cinerea using dual culture technique on SDYA. In addition, we also performed to screening of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging activity compounds from liquid culture filtrates of entomopathogenic fungi and investigate to it’s anticancer activity. As results, 12 isolates, 6 isolates and 25 isolates showing of these fungal metabolites produced antibacterial, antifungal and radicals scavenging activity compounds, respectively. The preferential antimicrobial and radical scavenging activities give evidence that these entomopathogenic fungal metabolites might be useful as a source for plant pathogen control and pharmaceutical interests.

The bulb mite (Rhizoglyphus echinopus) damages garlic, shallot and onion in the bulbs, corms and tubers. It has recently become a serious problem because of the continuous use of acaricides resulting in resistance among bulb mite population. Thus, there is need to find alternative control measures to suppress bulb mite population. Here, we report the screening result of pathogenic fungi for the control of R. echinopus. Initial screenings were performed using 352 isolates of entomopathogenic fungi from Korea soils. As results, 15 isolates of acaropathogenic fungi showed the pathogenicity to bulb mite supporting fungal conidiation. These isolates were identified as 3 isolates of Metarhizium flavoviride var. pemphigi and 12 isolates of Metarhizium pingshaense by microscopic examination and genetic sequencing of the ITS region and elongation factor-1 alpha. Selected 15 isolates were tested for their virulence against adult R. echinopus and the thermotolerance and the activity to UV-B irradiation of conidia. Additionally, the activities of chitinases and proteases produced by M. pingshaense were compared according to the medium. These acaropathogenic fungi would be considered promising for biological control of bulb mite.

Entomopathogenic fungi are natural pathogens of insects and contribute to the regulation of host insect populations in the environment. Several these fungi produce a wide range of secreted enzymes, secreted protein toxins and secondary metabolites to overcome host defenses and ultimately kill the host, and to defend host resources against competing pathogens and saprophytes. This study was performed to evaluate the antimicrobial activity of 207 entomopathogenic fungi form Korea soils against plant pathogenic bacteria Ralstonia solanacearum and plant pathogenic fungi Botrytis cinerea using dual culture technique on SDYA. As results, twelve isolates (5.7%) and six isolates (2.8%) showing the greatest inhibition against R. solanacearum and B. cinerea, respectively. The culture supernatant of these selected isolates completely suppressed the growth of the pathogen, indicating that suppression was due to the presence of antimicrobial compound in the culture filtrate. The stability test of the culture filtrate showed that the antimicrobial component was heat stable and not protein. These entomopathogenic fungal metabolites may be a good feature to be used in the development of a new biocontrol method of R. solanacearum and B. cinerea.

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to meet a multi-parellel process. We have developed a novel recombinant bacmid, bEasyBm that enabling easy and fast generation of pure recombinant virus without any purification step. In the bEasyBm, attR recombination sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus early promoter. Therefore, only when the barnase gene was replaced to gene of interest, the bEasyBm could replicate in host insect cells. When the bEasyBm was transposed with pDualBac-EGFP and pDualBac-LUC respectively, there were no non-recombinant backgrounds were detected from unpurified BmEasy-EGFP or BmEasy-LUC stocks. In addition, the resulting recombinant virus, BmEasy-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, BmEGFP, which was constructed using bBmGOZA system. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established.

Varieties of Bacillus thuringiensis (Bt) crystal proteins, Cry proteins, have so far been found as one of the most successful biological control agents which are safe to natural environments for a long time. Recently, cry genes encoding these Cry proteins have been widely applied for construction of transgenic crops resistant to pest insects. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. To construct novel cry genes with improved insecticidal activity, we randomly mutated these 23 amino acid sequences by in vitro muti site-directed mutagenesis, resulting in totally 24 mutant cry genes. For further characterization, these mutant cry genes were expressed as a fusion protein with polyhedrin using baculovirus expression system. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. In the further study, we plan to investigate their insecticidal activity against Plutella xylostella, S. exigua and O. furnacalis larvae.